The role of different levels of costimulation in conjunction with signal 1 in the activation of memory CD8+ T cells remains elusive. In this study, we demonstrate, in a mouse model with the influenza nucleoprotein epitope NP68, that mouse early memory (effector/memory) CD8+ T cells that were generated with high levels of costimulation have reduced cytotoxic T lymphocyte (CTL) functionality compared with those that were generated with low levels of costimulation. This reduction is associated with increased phosphorylation of the negative regulatory site 292 on Zap70 and a decrease in granzyme B levels. Furthermore, we show that enhanced costimulation reduces proliferation and cytokine production of effector/memory CD8+ T cells in response to intermediate and weak T-cell receptor (TCR) stimulation, in contrast to previously described positive effects of costimulation on naive CD8+ T cells. This effect is associated with the expression of ICAM-1 on APCs. Together, our results indicate that enhanced costimulation can lead to reduced functionality in effector/memory CD8+ T cells. This compromised effector function of effector/memory CD8+ T cells in response to high levels of costimulation can have important implications for designing immunotherapeutic strategies to enhance immune responses. In other studies we have demonstrated, for the first time, that murine T regulatory cells (Treg cells) in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4+CD25+FoxP3+GITRhighCD27lowCD62L. The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase. It is also demonstrated that tumor-infiltrating Treg cells have impaired TCR-mediated signaling and calcium influx. Based on these findings, this study supports the hypothesis that (1) tumor-infiltrating Treg cells lose functionality due to their diminished ability to become effectively activated and (2) intratumoral accumulation of Treg cells may compensate for the impaired functionality, thus maintaining immune tolerance to the tumor.